Formulations of tegavivint and related compounds

ABSTRACT

Formulations of tegavivint and related compounds, methods of making such formulations and methods of treating various conditions utilizing such formulations.

FIELD OF THE INVENTION

The present invention relates generally to formulations of tegavivintand related compounds, methods of making such formulations and methodsof treating various conditions utilizing such formulations.

BACKGROUND OF THE INVENTION

Cancer is the second leading cause of death in the United States. Itpresents complex challenges for the development of new therapies. Canceris characterized by the abnormal growth of malignant cells that haveundergone a series of genetic changes that lead to growth of tumor massand metastatic properties.

Beta-catenin (β-catenin) is part of a complex of proteins thatconstitute adherens junctions (AJs). AJs are necessary for the creationand maintenance of epithelial cell layers by regulating cell growth andadhesion between cells. β-catenin also anchors the actin cytoskeletonand may be responsible for transmitting the contact inhibition signalthat causes cells to stop dividing once the epithelial sheet iscomplete.

Wnt/β-catenin pathway has been shown to play a role in cancer. Aberrantβ-catenin signaling plays an important role in tumorigenesis. Inparticular, colorectal cancer is estimated to have greater than 80%mutations in the β-catenin pathway, leading to unregulated oncogenicsignaling. Aberrant β-catenin signaling has been shown to be involved invarious cancer types, including but not limited to, melanoma, breast,lung, colon, liver, gastric, myeloma, multiple myeloma, chronicmyelogenous leukemia, chronic lymphocytic leukemia, T-cell non-Hodgkinlymphomas, colorectal and acute myeloid leukemia (AML) cancers. Further,aberrant Wnt/β-catenin signaling has been found in a large number ofother disorders, including osteoporosis, osteoarthritis, polycystickidney disease, diabetes, schizophrenia, vascular disease, cardiacdisease, hyperproliferative disorders, neurodegenerative diseases, andfibrotic diseases including but not limited to idiopathic pulmonaryfibrosis (IPF), Dupuytren's contracture, Nonalcoholic steatohepatitis(NASH), and others. Myeloproliferative neoplasms (MPNs) are a closelyrelated group of hematological malignancies in which the bone marrowcells that produce the body's blood cells develop and functionabnormally. The three main myeloproliferative neoplasms are PolycythemiaVera (PV), Essential Thrombocythemia (ET) and Primary Myelofibrosis(PMF). A gene mutation in JAK2 is present in most PV patients and 50% ofET and PMF patients. The beta catenin pathway is activated in MPN inmany cases and required for survival of these cells.

Tegavivint and related compounds are described, for example, in U.S.Pat. No. 8,129,519. Tegavivint has the following structural formula:

The molecular formula of tegavivint is C₂₈H₃₆N₄O₆S₂.

The molecular mass of tegavivint is 588.20763 amu.

There is a need in the art to provide stable, readily bioavailableformulations of tegavivint and related compounds, wherein theformulations allow administration via different routes ofadministration, including but not limited to, parenteral and viainhalation, and are stable to be suitable for a clinical study andtreatment of various diseases which are treatable with tegavivint.

SUMMARY OF THE INVENTION

It has been very challenging and difficult to develop a stable,non-toxic formulation of tegavivint. A large number of formulations weredeveloped and tested; however, they had poor bioavailability and/orproved unstable upon storage, and/or turned to be highly toxic. Theseformulations include microemulsions, solid suspensions, liposome-basedformulations, various oral formulations, and IV formulations.

The inventors have unexpectedly and surprisingly discovered that ananosuspension of tegavivint works, wherein the nanosuspension comprisesa surfactant and wherein the particles of tegavivint have an effectiveD50 of less than or equal to 500 nm and D90 of less than or equal to 1.0micrometer (μm) when measured using laser diffraction. It has also beendiscovered that a particularly preferred concentration of tegavivint is10-25 mg/ml, most preferably 25 mg/ml; a preferred surfactant is apoloxamer surfactant (preferably, Poloxamer 188), preferably at aconcentration of 0.625%; and that the nanosuspension should preferablyinclude a polyol, and more preferably sorbitol.

The most preferred formulation, therefore, is a composition thatcomprises tegavivint at 25 mg/ml; Poloxamer 188 at 0.625% and 10%sorbitol, wherein tegavivint is in the form of a nanosuspensioncomprising particles of tegavivint, and wherein the particles have aneffective D50 of less than or equal to 500 nm and D90 of less than orequal to 1.0 micrometer (μm) when measured using laser diffraction.

Thus, in one embodiment, the invention provides a compositioncomprising:

a) particles of a compound of formula I

wherein R_(A) is hydrogen, R₇ and R₈ are independently selected from Hand SO₂NR₃R₄, wherein one of R₇ and R₈ is hydrogen and wherein NR₁R₂ andNR₃R₄ are independently 6- to 15-membered heterocycloalkyl containingone nitrogen in the ring, or a pharmaceutically acceptable salt, ester,amide, stereoisomer or geometric isomer thereof; and

b) a surfactant;

wherein the particles have an effective D50 of less than or equal to 500nm and D90 of less than or equal to 1.0 micrometer (μm) when measuredusing laser diffraction.

In some embodiments, the effective average particle size of thecompounds is about 4900 nm, about 4800 nm, about 4700 nm, about 4600 nm,about 4500 nm, about 4400 nm, about 4300 mm, about 4200 nm, about 4100nm, about 4 microns, about 3900 nm, about 3800 nm, about 3700 nm, about3600 nm, about 3500 nm, about 3400 mm, about 3300 nm, about 3200 nm,about 3100 nm, about 3 microns, about 2900 mm, about 2800 nm, about 2700nm, about 2600 nm, about 2500 nm, about 2400 nm, about 2300 nm, about2200 nm, about 2100 nm, about 2000 nm, about 1900 nm, about 1800 nm,about 1700 nm, about 1600 nm, about 1500 nm, about 1400 nm, about 1300nm, about 1200 nm, about 1100 nm, about 1000 nm, about 900 nm, about 800nm, about 700 nm, about 600 nm, about 500 nm, about 400 nm, or about 300nm.

Further, in some embodiments, the effective average particle size of thecompounds is less than 900 nm, more preferably less than 500 nm, andeven more preferably, less than 300 nm.

In a preferred embodiment, the surfactant is a poloxamer surfactant.

In another preferred embodiment, the poloxamer surfactant is Poloxamer188.

In a preferred embodiment, the particulate composition further comprisesa stabilizer.

In a preferred embodiment, the stabilizer is selected from the groupconsisting of a sugar, a polyol, a polysorbate surfactant andpolyvinylpyrrolidone (PVP).

In another preferred embodiment, the sugar is selected from the groupconsisting of sucrose and/or trehalose.

In a preferred embodiment, the polyol comprises sorbitol and/ormannitol.

In one embodiment, the concentration of the compound in the providedcompositions is between about 1 mg/ml and about 100 mg/ml, morepreferably between about 10 mg/ml and about 50 mg/ml, more preferablybetween about 10 mg/ml and about 25 mg/ml and even more preferably about25 mg/ml.

In one embodiment, the compositions of the invention are prepared bymilling.

In another embodiment, the compositions of the invention are prepared byLyoCell technology. U.S. Pat. No. 7,713,440 describes the LyoCelltechnology. The contents of U.S. Pat. No. 7,713,440 are herebyincorporated by reference in its entirety.

In another embodiment, the compositions of the invention can be preparedby a dry milling approach such as that described in U.S. Pat. No.8,808,751. The contents of U.S. Pat. No. 8,808,751 are herebyincorporated by reference in its entirety. By proper selection ofmilling media and suitable grinding compounds, it is possible togenerate a nanoparticulate composition from conventional drug substanceparticles and to prevent agglomeration of the small particles created inthe dry milling apparatus.

In yet another embodiment, the compositions of the invention can beprepared by a process utilizing human serum albumin as a carrier, suchas a process described in U.S. Pat. No. 6,537,579. The contents of U.S.Pat. No. 6,537,579 are hereby incorporated by reference in its entirety.This process may be particularly suited for making nanoparticulatecompositions of poorly water-soluble compounds. Compositions created bysuch a process may allow for effective administration of biologicallyactive compounds that are poorly water-soluble.

In another embodiment, nanoparticulate compositions containing polymerssuch as poly(DL-lactide-co-glycolide) are able to deliver poorly solublebiologically active compounds. As shown in U.S. Pat. No. 5,543,158,these compositions can be designed to be long-acting vehicles. Thecontents of U.S. Pat. No. 5,543,158 are hereby incorporated by referencein its entirety.

In another embodiment, compositions of the invention can be prepared aspolymeric micelles which have been successful in improving thesolubility of biologically active compounds. A marketed product usingthis technology, Genexol-PM, incorporates the anti-cancer drugpaclitaxel and was approved in South Korea in 2007.

In one embodiment, the invention provides a process of preparing acomposition comprising the following steps (a) through (c):

a) mixing particles of the compound of formula I

wherein R_(A) is hydrogen, R₇ and R₈ are independently selected from Hand SO₂NR₃R₄, wherein one of R₇ and R₈ is hydrogen and wherein NR₁R₂ andNR₃R₄ are independently 6- to 15-membered heterocycloalkyl containingone nitrogen in the ring,or a pharmaceutically acceptable salt, ester, amide, stereoisomer orgeometric isomer thereof;

-   -   with a surfactant and an acceptable carrier to produce a        suspension;

b) using a roller mill or high energy mill to mill the suspension ofstep (a); and

c) adding a polyol to the particles of step (b).

In one embodiment, the acceptable carrier is a liquid carrier (e.g.,water).

In one embodiment, the suspension is an aqueous suspension.

In another embodiment, the process of preparing a composition comprisesthe following steps (a) through (b):

a) mixing particles of the compound of formula I

wherein R_(A) is hydrogen, R₇ and R₈ are independently selected from Hand SO₂NR₃R₄, wherein one of R₇ and R₈ is hydrogen and wherein NR₁R₂ andNR₃R₄ are independently 6- to 15-membered heterocycloalkyl containingone nitrogen in the ring,or a pharmaceutically acceptable salt, ester, amide, stereoisomer orgeometric isomer thereof;

-   -   with a surfactant, a polyol and an acceptable carrier to produce        a suspension; and

b) using a roller mill or high energy mill to mill the suspension ofstep (a).

In one embodiment, the acceptable carrier is a liquid carrier (e.g.,water).

In one embodiment, the suspension is an aqueous suspension.

In a preferred embodiment, the compositions of the invention exhibitlong term stability.

In a preferred embodiment, the compositions of the invention arenanoparticulate compositions.

In a preferred embodiment, the compound of formula I in the compositionsof the invention has the following structure:

or a pharmaceutically acceptable salt, ester, amide, stereoisomer orgeometric isomer thereof.

The compound having the formula above is also known as tegavivint(BC2059).

In one embodiment, the compositions of the invention may be formulated:(a) into a dosage form selected from the group consisting of tablets,and capsules; (b) into a dosage form selected from the group consistingof controlled release formulations, fast melt formulations, delayedrelease formulations, extended release formulations, pulsatile releaseformulations, and mixed immediate release and controlled releaseformulations; (c) into a dosage form suitable for inhalation orparenteral administration, including intramuscular, subcutaneous,intravenous and intradermal injection; (d) any combination of (a), (b)and (c).

The compositions of the invention can further comprise one or morepharmaceutically acceptable excipients, carriers, or a combinationthereof.

In another embodiment, the invention provides a method of preventing,treating or ameliorating cancer or tumor metastasis in a mammal in needthereof comprising administering to said mammal an effective amount ofthe compositions of the invention.

The method of administering is not limited to any specific route ofadministration, and includes, but is not limited to, intravenous,parenteral, oral, inhalation (including aerosolized delivery), buccal,intranasal, rectal, intra-lesional intraperitoneal, intradermal,transdermal, subcutaneous, intra-arterial, intracardiac,intraventricular, intracranial, intratracheal, intrathecaladministration, intramuscular injection, intravitreous injection, andtopical application methods.

In another embodiment, the method of preventing, treating orameliorating cancer or tumor metastasis in a mammal in need thereof caninclude administering an additional anti-cancer agent and/or cancertherapy (for example, cancer vaccines, anti-cancer adoptive celltherapies and radio therapies).

In one embodiment, the additional anti-cancer agent is selected from thegroup consisting of antimitotic agents, antimetabolite agents, HDACinhibitors, proteosome inhibitors, immunotherapeutic agents, FLT-3 EGFR,MEK, PI3K and other protein kinase inhibitors, LSD1 inhibitors, and WNTpathway inhibitors, alkylating agents and DNA repair pathway inhibitors,anti-hormonal agents, anti-cancer antibodies, and other cytotoxicchemotherapy agents.

In another embodiment, the invention provides a method of treatingand/or preventing a fibrotic disease in a mammal in need thereofcomprising administering to said mammal an effective amount of thecompositions of the invention.

In a preferred embodiment, the fibrotic disease is selected from thegroup consisting of pulmonary fibrosis, Dupuytren's contracture,scleroderma, systemic sclerosis, scleroderma-like disorders, sinescleroderma, liver cirrhosis, interstitial pulmonary fibrosis, keloids,chronic kidney disease, chronic graft rejection, and otherscarring/wound healing abnormalities, post-operative adhesions, andreactive fibrosis.

In one embodiment, the method of treating and/or preventing a fibroticdisease in a mammal in need thereof can include administering anadditional anti-fibrotic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of Particle Size Distribution (PSD) of one of theinventive formulations.

FIG. 2 is a graph of PSD of another one of the inventive formulations.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. Synonyms for certain termsare provided. A recital of one or more synonyms does not exclude the useof other synonyms. The use of examples anywhere in this specificationincluding examples of any terms discussed herein is illustrative only,and in no way limits the scope and meaning of the invention or of anyexemplified term. The invention is not limited to the variousembodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control.

The term “tegavivint” refers to a compound having the followingstructure:

The term “BC2059” is used interchangeably with “tegavivint.”

The term “long-term storage” or “long-term stability” is understood tomean that the pharmaceutical composition can be stored for three monthsor more, for six months or more, and preferably for one year or more.Long term storage is also understood to mean that the pharmaceuticalcomposition is stored at 2-8° C. or at room temperature 15-25° C.

The term “stable” or “stabilized” with respect to long-term storage isunderstood to mean that active ingredient contained in thepharmaceutical compositions does not lose more than 20%, or morepreferably 15%, or even more preferably 10%, and most preferably 5% ofits activity relative to activity of the composition at the beginning ofstorage.

The term “mammal” includes, but is not limited to, a human.

The term “pharmaceutically acceptable carrier” refers to a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material,formulation auxiliary, or excipient of any conventional type. Apharmaceutically acceptable carrier is non-toxic to recipients at thedosages and concentrations employed and is compatible with otheringredients of the formulation.

The term “treatment” refers to any administration or application ofremedies for disease in a mammal and includes inhibiting the disease,arresting its development, relieving the disease (for example, bycausing regression, or restoring or repairing a lost, missing, ordefective function) or stimulating an inefficient process. The termincludes obtaining a desired pharmacologic and/or physiologic effect andcovering any treatment of a pathological condition or disorder in amammal. The effect may be prophylactic in terms of completely orpartially preventing a disorder or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disorder and/oradverse effect attributable to the disorder. It includes (1) preventingthe disorder from occurring or recurring in a subject who may bepredisposed to the disorder but is not yet symptomatic, (2) inhibitingthe disorder, such as arresting its development, (3) stopping orterminating the disorder or at least its associated symptoms, so thatthe host no longer suffers from the disorder or its symptoms, such ascausing regression of the disorder or its symptoms, for example, byrestoring or repairing a lost, missing or defective function, orstimulating an inefficient process, or (4) relieving, alleviating orameliorating the disorder, or symptoms associated therewith, whereameliorating is used in a broad sense to refer to at least a reductionin the magnitude of a parameter, such as inflammation, pain and/or tumorsize.

The term “therapeutically effective amount” refers to an amount which,when administered to a living subject, achieves a desired effect on theliving subject. For example, an effective amount of the compositions ofthe invention for administration to the living subject is an amount thatprevents and/or treats any of the diseases mediated via theWnt/β-catenin pathway. The exact amount will depend on the purpose ofthe treatment and will be ascertainable by one skilled in the art usingknown techniques. As is known in the art, adjustments for systemicversus localized delivery, age, body weight, general health, sex, diet,time of administration, drug interaction and the severity of thecondition may be necessary, and will be ascertainable with routineexperimentation by those skilled in the art.

The term “composition” or “formulation” refers to a mixture that usuallycontains a carrier, such as a pharmaceutically acceptable carrier orexcipient that is conventional in the art and which is suitable foradministration into a subject for therapeutic, diagnostic, orprophylactic purposes. For example, compositions for oral administrationcan form solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations, oral rinses or powders. The terms “composition,”“pharmaceutical composition” and “formulation” are used interchangeably.

The term “nanoparticulate composition” refers to compositions whereinall, or almost all of the particles are less than 1000 nM.

Compositions of the Invention

In one embodiment, the invention provides a composition comprising:

a) particles of a compound of formula I

wherein R_(A) is hydrogen, R₇ and R₈ are independently selected from Hand SO₂NR₃R₄, wherein one of R₇ and R₈ is hydrogen and wherein NR₁R₂ andNR₃R₄ are independently 6- to 15-membered heterocycloalkyl containingone nitrogen in the ring,or a pharmaceutically acceptable salt, ester, amide, stereoisomer orgeometric isomer thereof; and

b) a surfactant;

wherein the particles have an effective D50 of less than or equal to 500nm and D90 of less than or equal to 1.0 micrometer (μm) when measuredusing laser diffraction.

D50 is also known as median diameter of particle size distribution. Itrefers to the value of the particle diameter at 50% in the cumulativedistribution. In other words, when D50 value is less than or equal to500 nm, it means that 50% of the particles are less than 500 nm indiameter.

D90 refers to the percentage of the particles under the reportedparticle size. In other words, when D90 value is less than or equal to1.0 μm, it means that 90% of the particles are less than 1.0 μm indiameter.

In some embodiments, the effective average particle size of thecompounds is about 4900 nm, about 4800 nm, about 4700 nm, about 4600 nm,about 4500 nm, about 4400 nm, about 4300 mm, about 4200 nm, about 4100nm, about 4 microns, about 3900 nm, about 3800 nm, about 3700 nm, about3600 nm, about 3500 nm, about 3400 mm, about 3300 nm, about 3200 nm,about 3100 nm, about 3 microns, about 2900 mm, about 2800 nm, about 2700nm, about 2600 nm, about 2500 nm, about 2400 nm, about 2300 nm, about2200 nm, about 2100 nm, about 2000 nm, about 1900 nm, about 1800 nm,about 1700 nm, about 1600 nm, about 1500 nm, about 1400 nm, about 1300nm, about 1200 nm, about 1100 nm, about 1000 nm, about 900 nm, about 800nm, about 700 nm, about 600 nm, about 500 nm, about 400 nm, or about 300nm.

Further, in some embodiments, the effective average particle size of thecompounds is less than 900 nm, more preferably less than 500 nm, andeven more preferably, less than 300 nm.

In a preferred embodiment, the surfactant is a poloxamer surfactant.

In another preferred embodiment, the poloxamer surfactant is Poloxamer188.

In a preferred embodiment, the composition further comprises astabilizer.

In a preferred embodiment, the stabilizer is selected from the groupconsisting of a sugar, a polyol, a polysorbate surfactant andpolyvinylpyrrolidone (PVP).

In another preferred embodiment, the sugar is selected from the groupconsisting of sucrose and/or trehalose.

In a preferred embodiment, the polyol comprises sorbitol and mannitol.

In one embodiment, the concentration of the compound in the providedcompositions is between about 1 mg/ml and about 100 mg/ml, morepreferably between about 10 mg/ml and about 50 mg/ml, more preferablybetween about 10 mg/ml and about 25 mg/ml and even more preferably about25 mg/ml.

A particularly preferred concentration of tegavivint is 10-25 mg/ml,most preferably 25 mg/ml; a preferred surfactant is a poloxamersurfactant (preferably, Poloxamer 188), preferably at a concentration of0.625%; and the nanosuspension preferably includes a polyol, and morepreferably sorbitol.

The most preferred formulation, therefore, is a nanosuspension thatcomprises tegavivint at 25 mg/ml; Poloxamer 188 at 0.625% and 10%sorbitol.

In one embodiment, the compositions of the invention are prepared bymilling, preferably wet milling.

In one embodiment, the invention provides a process of preparing acomposition comprising the following steps (a) through (c):

a) mixing particles of the compound of formula I

wherein R_(A) is hydrogen, R₇ and R₈ are independently selected from Hand SO₂NR₃R₄, wherein one of R₇ and R₈ is hydrogen and wherein NR₁R₂ andNR₃R₄ are independently 6- to 15-membered heterocycloalkyl containingone nitrogen in the ring,or a pharmaceutically acceptable salt, ester, amide, stereoisomer orgeometric isomer thereof;

-   -   with a surfactant and an acceptable carrier to produce a        suspension;

b) using a roller mill or high energy mill to mill the suspension ofstep (a); and

c) adding a polyol to the particles of step (b).

In one embodiment, the acceptable carrier is a liquid carrier (e.g.,water).

In one embodiment, the suspension is an aqueous suspension.

In another embodiment, the process of preparing a composition comprisesthe following steps (a) through (b):

a) mixing particles of the compound of formula I

wherein R_(A) is hydrogen, R₇ and R₈ are independently selected from Hand SO₂NR₃R₄, wherein one of R₇ and R₈ is hydrogen and wherein NR₁R₂ andNR₃R₄ are independently 6- to 15-membered heterocycloalkyl containingone nitrogen in the ring,or a pharmaceutically acceptable salt, ester, amide, stereoisomer orgeometric isomer thereof;

-   -   with a surfactant, a polyol and an acceptable carrier to produce        a suspension; and

b) using a roller mill or high energy mill to mill the suspension ofstep (a).

In one embodiment, the acceptable carrier is a liquid carrier (e.g.,water).

In one embodiment, the suspension is an aqueous suspension.

In another embodiment, the compositions of the invention are prepared byLyoCell technology. U.S. Pat. No. 7,713,440 describes the LyoCelltechnology. The contents of U.S. Pat. No. 7,713,440 are herebyincorporated by reference in its entirety.

In another embodiment, the compositions of the invention can be preparedby a dry milling approach such as that described in U.S. Pat. No.8,808,751. The contents of U.S. Pat. No. 8,808,751 are herebyincorporated by reference in its entirety. By proper selection ofmilling media and suitable grinding compounds, it is possible togenerate a nanoparticulate composition from conventional drug substanceparticles and to prevent agglomeration of the small particles created inthe dry milling apparatus.

In yet another embodiment, the compositions of the invention can beprepared by a process utilizing human serum albumin as a carrier, suchas a process described in U.S. Pat. No. 6,537,579. The contents of U.S.Pat. No. 6,537,579 are hereby incorporated by reference in its entirety.This process may be particularly suited for making nanoparticulatecompositions of poorly water-soluble compounds. Compositions created bysuch a process may allow for effective administration of biologicallyactive compounds that are poorly water-soluble.

In another embodiment, nanoparticulate compositions containing polymerssuch as poly(DL-lactide-co-glycolide) are able to deliver poorly solublebiologically active compounds. As shown in U.S. Pat. No. 5,543,158,these compositions can be designed to be long-acting vehicles. Thecontents of U.S. Pat. No. 5,543,158 are hereby incorporated by referencein its entirety.

In another embodiment, compositions of the invention can be prepared aspolymeric micelles which have been successful in improving thesolubility of biologically active compounds. A marketed product usingthis technology, Genexol-PM, incorporates the anti-cancer drugpaclitaxel and was approved in South Korea in 2007.

In a preferred embodiment, the compositions of the invention exhibitlong term stability.

In one embodiment, the compositions of the invention are nanoparticulatecompositions.

In a preferred embodiment, the compound of formula I in the compositionsof the invention has the following structure:

or a pharmaceutically acceptable salt, ester, amide, stereoisomer orgeometric isomer thereof.

This compound is also known as tegavivint.

The invention encompasses formulations including tegavivint and apharmaceutically acceptable salt, ester, amide, stereoisomer orgeometric isomer thereof.

Tegavivint solubility in water has been measured across the pH range of2 to 10 and was found to be <0.25 mcg/mL across the range.

In organic solvents, tegavivint has solubilities as shown: DMSO (334μg/mL), ethanol (260 μg/mL), methanol (299 μg/mL), acetone (1 mcg/mL),dichloromethane:ethanol (1:4) (1 mg/mL).

In one embodiment, the compositions of the invention may be formulated:(a) into a dosage form selected from the group consisting of tablets,and capsules; (b) into a dosage form selected from the group consistingof controlled release formulations, fast melt formulations, delayedrelease formulations, extended release formulations, pulsatile releaseformulations, and mixed immediate release and controlled releaseformulations; (c) into a dosage form suitable for inhalation orparenteral administration, including intramuscular, subcutaneous,intravenous and intradermal injection; (d) any combination of (a), (b)and (c).

The compositions of the invention can further comprise one or morepharmaceutically acceptable excipients, carriers, or a combinationthereof.

The pharmaceutically acceptable excipients used in the formulation ofthe present invention can act in more than one way. The role ofdispersant, for example, is principally to allow individual particles toremain separated, i.e. to minimize agglomeration. However, thisingredient might also impart changes to surface tension of theformulation, for instance, and might act to reduce viscosity.

The pharmaceutically acceptable excipients can be, for example, adispersion medium, a dispersion emulsifier, a dispersion enhancer, or acombination thereof.

Examples of the propellant include, but not limited to, HFA-134a (1, 1,1, 2-tetrafluoroethane), HFA-227 (1,1,1,2,3,3,3-heptafluoropropane), acombination thereof, etc.

The dispersion medium can be, for example, ethanol, propylene glycol,polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol400, glycerin, a combination thereof, etc.

The dispersion emulsifier (enhancer) can be, for example, H₂O, oleicacid, sodium lauryl sulfate, polyethylene glycol 1000, ammoniumalginate, potassium alginate, calcium stearate, glyceryl monooleate,polyoxyethylene stearates, emulsifying wax, polysorbate 20, polysorbate40, polysorbate 60, polysorbate 80, sorbitan monolaurate, sorbitanmonooleate, sorbitan monopalmitate, sorbitan monostearate, sorbitansesquioleate, sorbitan trioleate, poloxamer, a combination thereof, etc.

Examples of the dispersion enhancers include, but not limited to,polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80,carboxymethylcellulose sodium, hypromellose, ethylene glycol stearates,sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate,sorbitan monostearate, sorbitan sesquioleate, sorbitan trioleate,glyceryl monostearate, lecithin, meglumine, poloxamer, polyoxyethylenealkyl ethers, polyoxyl 35 castor oil, polyoxyethylene stearates,polyoxylglycerides, pyrrolidone, sorbitan esters, stearic acid, vitaminE polyethylene glycol succinate, polyethylene glycol 1000, povidone, acombination thereof, etc.

The compositions of the invention can be suitable for all routes ofadministration, including but not limited to, intravenous, parenteral,oral, inhalation (including aerosolized delivery), buccal, intranasal,rectal, intra-lesional intraperitoneal, intradermal, transdermal,subcutaneous, intra-arterial, intracardiac, intraventricular,intracranial, intratracheal, intrathecal administration, intramuscularinjection, intravitreous injection, and topical application methodsPharmaceutical compositions according to the invention may also compriseone or more binding agents, filling agents, lubricating agents,suspending agents, sweeteners, flavoring agents, preservatives, buffers,wetting agents, disintegrants, effervescent agents, and otherexcipients. Such excipients are known in the art.

Examples of filling agents are lactose monohydrate, lactose anhydrous,and various starches; examples of binding agents are various cellulosesand cross-linked polyvinylpyrrolidone, microcrystalline cellulose, suchas Avicel® PH101 and Avicel® PH102, microcrystalline cellulose, andsilicified microcrystalline cellulose (ProSolv SMCC™).

Suitable lubricants, including agents that act on the flowability of thepowder to be compressed, are colloidal silicon dioxide, such as Aerosil®200, talc, stearic acid, magnesium stearate, calcium stearate, andsilica gel.

Examples of sweeteners are any natural or artificial sweetener, such assucrose, xylitol, sodium saccharin, cyclamate, aspartame, and acsulfame.Examples of flavoring agents are Magnasweet® (trademark of MAFCO),bubble gum flavor, and fruit flavors, and the like.

Examples of preservatives are potassium sorbate, methylparaben,propylparaben, benzoic acid and its salts, other esters ofparahydroxybenzoic acid such as butylparaben, alcohols such as ethyl orbenzyl alcohol, phenolic compounds such as phenol, or quarternarycompounds such as benzalkonium chloride.

Suitable diluents include pharmaceutically acceptable inert fillers,such as microcrystalline cellulose, lactose, dibasic calcium phosphate,saccharides, and/or mixtures of any of the foregoing. Examples ofdiluents include microcrystalline cellulose, such as Avicel® PH101 andAvicel® PH102; lactose such as lactose monohydrate, lactose anhydrous,and Pharmatose® DCL21; dibasic calcium phosphate such as Emcompress®;mannitol; starch; sorbitol; sucrose; and glucose.

Suitable disintegrants include lightly crosslinked polyvinylpyrrolidone, corn starch, potato starch, maize starch, and modifiedstarches, croscarmellose sodium, cross-povidone, sodium starchglycolate, and mixtures thereof.

Examples of effervescent agents are effervescent couples such as anorganic acid and a carbonate or bicarbonate. Suitable organic acidsinclude, for example, citric, tartaric, malic, fumaric, adipic,succinic, and alginic acids and anhydrides and acid salts. Suitablecarbonates and bicarbonates include, for example, sodium carbonate,sodium bicarbonate, potassium carbonate, potassium bicarbonate,magnesium carbonate, sodium glycine carbonate, L-lysine carbonate, andarginine carbonate. Alternatively, only the sodium bicarbonate componentof the effervescent couple may be present.

In a preferred embodiment, the compound of formula I has the followingstructure:

or a pharmaceutically acceptable salt, ester, amide, stereoisomer orgeometric isomer thereof.

In another embodiment, the invention provides a method of preventing,treating or ameliorating cancer or tumor metastasis in a mammal in needthereof comprising administering to said mammal an effective amount ofthe compositions of the invention.

In another embodiment, the method of preventing, treating orameliorating cancer or tumor metastasis in a mammal in need thereof caninclude administering an additional anti-cancer agent and/or cancertherapy (for example, cancer vaccines, anti-cancer adoptive celltherapies and radio therapies).

In one embodiment, the additional anti-cancer agent is selected from thegroup consisting of antimitotic agents, antimetabolite agents, HDACinhibitors, proteosome inhibitors, immunotherapeutic agents, FLT-3 EGFR,MEK, PI3K and other protein kinase inhibitors, LSD1 inhibitors, and WNTpathway inhibitors, alkylating agents and DNA repair pathway inhibitors,anti-hormonal agents, anti-cancer antibodies, and other cytotoxicchemotherapy agents.

In another embodiment, the invention provides a method of treatingand/or preventing a fibrotic disease in a mammal in need thereofcomprising administering to said mammal an effective amount of thenanoparticulate compositions of the invention.

In a preferred embodiment, the fibrotic disease is selected from thegroup consisting of pulmonary fibrosis, Dupuytren's contracture,scleroderma, systemic sclerosis, scleroderma-like disorders, sinescleroderma, liver cirrhosis, interstitial pulmonary fibrosis, keloids,chronic kidney disease, chronic graft rejection, and otherscarring/wound healing abnormalities, post-operative adhesions, reactivefibrosis.

The present invention is more particularly described in the followingexamples that are intended as illustrative only, since manymodifications and variations therein will be apparent to those skilledin the art. In the following examples it should be understood thatweight percentages of various ingredients are expressed as w/vpercentages.

EXAMPLES OF THE INVENTION

It was very challenging and difficult to arrive at a formulation oftegavivint that worked: i.e., was stable and not toxic.

The formulation that worked turned out to be a nanosuspension oftegavivint, wherein the nanosuspension comprises a surfactant andwherein the particles of tegavivint have an effective D50 of less thanor equal to 500 nm and D90 of less than or equal to 1.0 micrometer (μm)when measured using laser diffraction. It has also been discovered thata particularly preferred concentration of tegavivint is 10-25 mg/ml,most preferably 25 mg/ml; a preferred surfactant is a poloxamersurfactant (preferably, Poloxamer 188), preferably at a concentration of0.625%; and that the nanosuspension should preferably include a polyol,and more preferably sorbitol.

The Examples section first describes multiple experiments to formulatetegavivint that ultimately failed for various reasons. Then, the sectiondescribes a milling feasibility experiment which demonstrated thattegavivint could be roller milled when suspended in aqueous solutionswith various dispersants. However, even when roller milled, multipleformulations of tegavivint were still unsuccessful.

Finally, it describes successful experiments which involved the claimednanosuspension of tegavivint.

Unsuccessful Experiments Example 1 A Microemulsion Formulation ofTegavivint was Very Toxic

A microemulsion formulation of tegavivint was developed, wherein theformulation contained 20 mg/ml BC2059, 10% Tween (polysorbate 80), 30%ethanol, 50% propylene glycol (PG) and 10% D-α-tocopherol polyethyleneglycol 1000 succinate.

Although good stability of the formulation was observed, the formulationwas extremely toxic to rodents and therefore not pursued further.

Example 2

Liposome-Based Formulations were Unstable

Based on preliminary studies, two liposome formulations of BC2059 werechosen as the leads for scale-up at 100 ml and stability evaluation.

The first was 100% ePC formulation with 15:1 lipid to drug ratio.

The other lead formulation included 80:20% ePC: LysoPC lyposomes at 10:1lipid to drug ratio.

Both of these formulations proved to be unstable upon storage at 5° C.(precipitation was observed). Furthermore, the formulations were alsounstable upon freezing.

Example 3

Oral and IV Formulations were Unsuccessful

An oral formulation of tegavivint containing soy lecithin, PEG200,PEG400, PG, and TPGS was initially elected as the lead oral formulation.However, this lead oral formulation showed poor bioavailability in thedog study and therefore was not pursued.

Upon further screening, an IV based formulation was elected as the nextlead. This IV formulation comprised an oil phase (vegetable oil andPolysorbate 80 (PS80) as solubilizers) and soy lecithin as theemulsifier. The formulation had the following ingredients (all numbersare weight %):

BC2059: 1%; PS80: 10%; Miglyol 812: 12%; soy lecithin (LIPOID S-100):12%; propylene glycol (PG): 50%; deionized water: to qs.

This formulation showed potential for filtration through 0.2 micron withminimal loss along with good physical and chemical stability. However,due to high toxicity in the rodent study, this formulation was notpursued further.

Experiments Involving Nanosuspensions of Tegavivint Example 4 MillingFeasibility

First, it was determined whether milling is feasible in principle. Theexperiment demonstrated that it is.

Milling feasibility was initiated by roller milling laboratory-scalebatches of tegavivint suspended at 5% (50 mg/mL) in aqueous solutionsthe following dispersants, selected for their suitability forintravenous administration:

-   -   Polysorbate 20 (0.5%)    -   Poloxamer 188 (0.5%)    -   Polyvinylpyrrolidone, K17 (1%)    -   Polyvinylpyrrolidone, K17 (1%) and sodium deoxycholate (0.25%)    -   Lecithin (1%)

5-mL test suspensions were milled each with a media charge ofapproximately 10 mL of 0.5-mm diameter yttria-stabilized zirconia (YTZ)milling media and were sampled periodically for particle-sizedistribution analysis by laser diffraction. After twelve hours ofmilling, only the poloxamer and the polyvinylpyrrolidone suspensionsshowed the production of a uniform nanoparticulate dispersion, with thelecithin suspension showing no appreciable size reduction, thepolysorbate suspension exhibiting caking of the BC2059 inside of themilling container, and the polyvinylpyrrolidone/sodium deoxycholatesuspension showing high aspect-ratio crystals. Finished test suspensionswere allowed to stand at uncontrolled ambient conditions for four daysfor informal particle-size stability. All tested suspensions showed somedegree of particulate growth, with particle elongation similar to whathad been seen initially in the polyvinylpyrrolidone/sodium deoxycholatesuspension.

To try to prevent crystal growth, preparations were made at 5% (50mg/mL) tegavivint using both poloxamer and polyvinylpyrrolidone (PVP) asdispersants and incorporating sucrose, sorbitol, and trehalose, each at10%. Milling and storage were done under similar conditions to theinitial feasibility experiment. All preparations milled down to ananosuspension, but none of the additives appeared to have a discernableeffect on crystal growth inhibition. For further work, poloxamer 188 waschosen as the primary dispersant.

Additional material was milled at 5% (50 mg/mL) in poloxamer 188. Tofacilitate an increase in scale, the poloxamer content was increasedfrom 1% to 1.5% to ensure a uniform nanosuspension. Millednanosuspension was diluted to produce a 2% (20 mg/mL) BC2059/0.6%poloxamer/0.9% sodium chloride formulation for use in initialpharmacokinetic work to be done by a third party. The remaining milledconcentrated material was reserved to test the effectiveness oflyophilization on preventing the apparent crystal growth.

This formulation was then tested in the experiment described in Example5.

Example 5 Lyophilization Feasibility

This experiment was supposed to determine if lyophilization is feasiblein principle. It showed that in principle, tegavivint can belyophilized.

The 5% (50 mg/mL) tegavivint poloxamer aqueous suspension was dilutedwith various potential cryoprotectant-containing diluents so that thefinal concentrations were 2% (20 mg/mL) tegavivint, 0.6% poloxamer, andthe following:

-   -   Sucrose (10%)    -   Mannitol (5%)    -   Sucrose (5%) and mannitol (2.5%)    -   Sorbitol (10%)    -   Sorbitol (5%) and mannitol (2.5%)    -   Trehalose (10%)    -   Trehalose (5%) and mannitol (2.5%)

5-mL serum vials were filled to 2 mL with each preparation andlyophilized at −40° C. and 100 mTorr pressure. The dried vials wereresuspended with purified water and analyzed for particle-sizedistribution. Of the systems tested, only the 10% sorbitol and the 10%trehalose resuspensions returned particle-size distributions that werecomparable to the pre-lyophilized suspension. Additional nanosuspensionwas milled, increasing the component concentrations to 10% (100 mg/mL)BC2059 and 3% poloxamer to increase milling efficiency and to facilitatelarger batch manufacture.

The following suspensions were prepared from the milled material forlow-temperature differential scanning calorimetry (DSC) analysis:

-   -   2% (20 mg/mL) BC2059 with 0.6% polysorbate and 10% sorbitol    -   2% (20 mg/mL) BC2059 with 0.6% polysorbate and 10% trehalose.

DSC analysis, performed from 25° C. to −40° C. and then back to 25° C.at a rate of 1° C. per minute, gave the following glass transitionvalues for the suspensions:

-   -   Sorbitol suspension: −18° C.    -   Trehalose suspension: −33° C.

The suspensions were lyophilized, with 2 mL fill in 5-mL vials, withprimary drying at −30° C./150 mTorr and with secondary drying at −16°C./550 mTorr. Informally, lyophilized samples were shown to bephysically stable, with reproducible uniform size distributions, for upto one week at ambient lab conditions. For subsequent work, sorbitol waschosen over trehalose because of both the higher glass-transitiontemperature and the greater availability of historical toxicity data onthe former.

A test batch was milled and lyophilized with primary drying at −24°C./250 mTorr and secondary drying at −16° C./500 mTorr to providematerials for an animal study. The milling was done at 20% (200 mg/mL)tegavivint with a poloxamer content of 5% to try to facilitate largerbatch sizes and to enhance milling efficiency. The dried formulationmeasured at about 1% water by Karl Fischer and showed adequateparticle-size stability after 24 hours when reconstituted with purifiedwater.

These formulations were then tested in the experiment described inExample 6.

Example 6 Nonclinical Toxicology/Pharmacokinetics Batch Production

The purpose of this experiment was to test the lyophilized formulationsof tegavivint.

Four sequentially prepared sub-batches of suspension, each representing15 g of tegavivint, were milled at the increased loading and wereextracted from the milling media using a diluent of 11.43% sorbitolaqueous solution to enhance the product yield and to result in asuspension of 2% (20 mg/mL) tegavivint/0.5% poloxamer/10% sorbitol.

Sub-batches were filled at 2 mL into 5-mL vials and lyophilized at thepreviously optimized conditions. Although some of the vials showed signsof meltback, likely due to the increase in batch scale, the driedmaterial resuspended readily into uniform nanosuspensions. The interimassay, PSD, and water results for each sub-batch of vials showedacceptable batch-to-batch agreement, so the four sets of vials werecombined and treated as a single batch for stability and animal studyuse. See Table 1 below.

TABLE 1 Sub- Assay D90 Water Batch (% LC) (um) (% w/w) 1 97.3 0.19 1.9 2103.1 0.18 1.5 3 111.3 0.18 1.1 4 105.9 0.19 1.1

During milling, one of the sub-batches failed because of stable foamproduction, which prevented further milling and resulted in permanentparticle aggregation. It was discarded and another batch was made toreplace it. In the production of the failing batch, a 250-mL serumbottle was used instead of a media bottle to facilitate PSD samplingduring milling. The foaming was attributed to the difference indimensions of the bottle which ostensibly allowed the entrainment ofair, resulting in the batch failure. During the extraction of allbatches, dark insoluble particulate was isolated from the milling media.This material was later analyzed by XRPD and found to be fusedaggregates of tegavivint.

At the one-month stability point, the composited batch exhibitedsignificant particle-size increase, attributed to aggregation ofpoloxamer rather than ripening or crystal growth of the API (drugsubstance). Attempts were made to determine a lab-suitable path by whichthe test articles could be salvaged for use. Samples were reconstituted,resealed, and heated to 50° C. for up to 3 hours without reducing theaggregates. Autoclaving the resuspended vials at 121° C. for 10 minutesusing a slow-release liquid cycle and allowing them to cool to ambientconditions returned an acceptable particle-size distribution.

As this kind of treatment did not present a suitable path forward, thedecision was made to re-formulate the product.

Example 7 Re-Formulating Compositions and Lyophilization UltimatelyFailed but Liquid Suspension Containing Poloxamer Appeared Promising

In this experiment, additional re-formulated compositions of tegavivintwere tested. Ultimately, lyophilization did not work but liquidsuspension (nanosuspension) showed promising results.

Feasibility-scale batches were made in some of the originally testeddispersants, but using the increased 20% (200 mg/mL) tegavivintconcentration, as this change might have made feasible a dispersant thathad not shown promise at 5% (5 mg/mL). The following dispersants weretested alongside a 5% poloxamer 188 control:

-   -   Polysorbate 20 (2%)    -   Polyvinylpyrrolidone (2%)    -   Polyvinylpyrrolidone (2%) and sodium deoxycholate (1%)    -   Polyvinylpyrrolidone (2%) and polysorbate 20 (2%)    -   Polyvinyl alcohol, partially hydrolyzed (5%)

After 12 hours of milling, the polysorbate preparation showed fastermilling than the control with good uniformity. The polyvinylpyrrolidonepreparation showed the presence of non-crystalline particles, possiblyaggregates or residuals of polyvinylpyrrolidone, which did notsignificantly affect the particle-size distribution measurements, butwhich were visible by light microscopy. The polyvinyl alcoholpreparation did not produce significant size reduction, likely due tothe viscosity of the dispersant. The two-component polyvinylpyrrolidonepreparations showed significant aggregates, but it was decided that thepolyvinylpyrrolidone/sodium deoxycholate preparation might prove to beuseful with additional development.

The polyvinylpyrrolidone and polysorbate 20 preparations, along with amodified polyvinylpyrrolidone (1%) and sodium deoxycholate (0.5%)suspensions were used in a lyophilization development experimentinvolving the following cryoprotectants:

-   -   Sorbitol (10%)    -   Sucrose (10%)    -   Trehalose (10%)    -   Mannitol (5%    -   Mannitol (5%)    -   Sorbitol (5%) and mannitol (2.5%)    -   Sucrose (5%) and mannitol (2.5%)

Lyophilization was done at −36° C./100 mTorr and −15° C./500 mTorr witha −15° C. annealing step. Upon resuspension, only the 10% sucrosepreparation gave suitable particle size recovery. Additional suspensionwas milled using the 1% polyvinylpyrrolidone/0.5% sodium deoxycholatepreparation, but with a citrate buffer included to maintain the pH ataround 7.0. Although the milled suspension preparation gelled reversiblyon standing, it was combined with the following cryoprotectants:

-   -   Sucrose (15%)    -   Sucrose (10%) at 25 mg/mL BC2059    -   Sorbitol (10%)    -   Lactose (5%)    -   Sucrose (5%) and sorbitol (5%)

Sucrose in higher concentrations relative to the API was shown toprovide the best particle-size protection and, although the formulationappeared to be susceptible to melt-back, an accelerated stability study,performed at 25° C./60% RH and 40° C./75 RH, showed that the formulationhad good physical stability over 4 weeks.

However, in dilution tests, the formulation was found to flocculate inthe saline diluent used in administration, and the pharmacokineticrelease of the polyvinylpyrrolidone/sodium deoxycholate formulation wassignificantly lower than that of the poloxamer formulation originallytested.

Nanosuspension Containing Poloxamer 188

200 mg/mL (20%) BC2059 was milled in poloxamer 188 and provided to athird party for lyophilization optimization. The suspension was combinedwith a series of cryoprotectants, listed in Table 2 below, that wereused in a lyophilization experiments. Initially, the 2.5% dextran/2.5%sorbitol preparation showed the most promising particle-size retentionupon reconstitution, however, after one month at 40 C/75% RH, the onlypreparation that had retained a nanosuspension was the undried control.

Accordingly, these tests indicated that lyophilization did not workunder the conditions evaluated. This finding also indicated thatsurprisingly the liquid suspension was more stable than had beenpreviously observed. The initial particle elongation was determined tobe an immediate and limited phenomenon, the possible result of initialover-saturation of the dispersant causing minor reprecipitation afterthe cessation of milling. See Table 2 below.

TABLE 2 Cryoprotectant System D90 Initial (um) D90 @ T1 M None, undried0.239 0.28 None, dried 23 61 10% sucrose 0.309 1.7 5% sucrose 7.539 2510% dextran 0.348 2.7 5% dextran 0.361 2.9 5% dextran + 5% sucrose 0.3528.8 2.5% dextran + 2.5% 0.29 18 sucrose 5% dextran + 5% sorbitol 1.02 402.5% dextran + 2.5% 0.987 24 sorbitol

Example 8 Irradiation Feasibility

Both of the developed formulation dispersant systems(polyvinylpyrrolidone/sodium deoxycholate and the poloxamer) were usedto determine the feasibility of terminal sterilization by irradiation.Samples of both were prepared for both irradiation feasibility. Samplesof both formulations were provided for a parallel pharmacokinetics (PK)study in laboratory animals, for which was also provided a diluentcontaining poloxamer 188 to be used with the polyvinylpyrrolidone/sodiumdeoxycholate formulation to determine if the bioavailability of the drugwas related to the poloxamer. Results of the PK study showed thatbioavailability surprisingly correlated with poloxamer content.

Frozen vials of both formulations were sent for irradiation. Both gammaand e-beam irradiation were tested at both 15 and 25 kGy. Vials were tobe processed under frozen conditions, but also at 5° C. as a worst-casescenario to simulate potential thawing during irradiation. Degradationwas independent of temperature but appeared to correlate with dose,regardless of the type of radiation.

However, initial particle-size testing, supported by subsequentstability data, showed extensive particle aggregation. Based onpreviously successful freeze/thaw testing, the aggregation wasattributed to irradiation; however, later freeze/thaw cycling on anothersuspension showed similar aggregation.

It was determined that frozen storage resulted in unpredictableaggregation and was not the choice to move forward with GLP batches. Thevials had been stored in a −20 C freezer prior to irradiation, andlikely exhibited differential freezing rates between vials at differentlocations on the shelf.

Example 9 Preclinical Production

The production of 25 mg/m L tegavivint nanosuspension was done usingbest-clean conditions, i.e., various controls and precautions were putin place to try to minimize microbial contamination, but withoutguarantee of sterility.

Preparations were made using sterile water for injection to minimize notonly microbial but also pyrogenic contamination. All excipients wereUSP/NF grade. All product contact supplies were either sterilized byautoclave, or, if not amenable to heating, sanitized with 70%isopropanol. All exposed preparations were performed in an ISO 5 qualitylaminar-flow hood using aseptic handling techniques. The API that wasused in the manufacture of the preclinical batches was gamma-irradiatedat 30 kGy prior to use.

Example 10 Rat Test Article Preparation

A 1,600-gram (nominal) batch of tegavivint suspension was prepared foradministration in a rat toxicology study.

Production began with a 200-gram batch of concentrated (200 mg/mL)BC2059 nanosuspension. 10 g of poloxamer 188 was dissolved in 150 gramsof water in a 250-mL serum bottle. 200 grams of YTZ milling media wereadded and the bottle stoppered and sealed. Given the small batch size,the entire assembly and poloxamer solution preparation was able to beautoclaved at 121 C for 15 minutes to minimize bioburden. 40 grams ofirradiated API (drug substance) was added and the bottle stoppered andsealed again. This preparation was rolled on a roller mill so that theangle of break of the cascading media was about 45 degrees, visuallydetermined.

Because of the tendency for the formulation to fail due to theentrainment of air, it was noted that the amount of milling media usedis about half of what would normally be used to process a 200-gram batchof suspension. The bottle used was also smaller than typical to minimizeheadspace. Milling was allowed to proceed over a weekend, and thesuspension was sampled via hypodermic needle through the septum.

The particle-size distribution had a D90 of 0.23 microns and wasdetermined to be sufficient to proceed to extraction, which was doneusing an autoclaved solution of 160 grams of sorbitol in 1240 gramswater and a glass pressure funnel containing a 60-micron sintered glassfrit. The extracted suspension was mixed and filled using apositive-displacement pipette set to 5.00 mL into autoclaved 10-mL glassvials. 295 vials were filled, stoppered, and sealed, representing a 92%yield. The batch was stored at 5° C. until use.

The nanosuspension appeared ready to be administered to rats.

Example 11 Pig Test Article Preparation

A 10,400-gram (nominal) batch of tegavivint was prepared foradministration in a pig toxicology study. Production began with a1,300-gram batch of concentrated (200 mg/mL) BC2059 nanosuspension. 65 gof poloxamer 188 was dissolved in 975 grams of water in a 2000-mL mediabottle. 1000 grams of YTZ milling media were rinsed and bagged forsterilization. The media and solution were autoclaved separately andcombined with 260 grams of sterilized API in the media bottle. Thispreparation was rolled on a roller mill so that the angle of break ofthe cascading media was about 45 degrees, visually determined. Millingwas allowed to proceed for a total of about three days, until theparticle-size distribution had a D90 of 0.33 microns and was determinedto be sufficient to proceed to extraction. Two aliquots of sorbitolsolution, made by dissolving 520 g of sorbitol in 4030 g of water, wereautoclaved and used to extract the milled suspension, similarly to whathad been done for the rat-study batch.

Difficulty was noted in extraction, as apparently unmilled or largerparticle size API had clogged the 60 micron filter frit, necessitatingthe removal of the media and the rinsing of the frit. The extractedsuspension was mixed and filled using a positive-displacement pipetteset to 10.0 mL into autoclaved 10-mL glass vials. 970 vials were filled,stoppered, and sealed, representing a 93% yield. The batch was stored at5° C. until use.

The nanosuspension appeared ready to be administered to pigs.

Example 12 Autoclaving Had no Significant Effect on Degradation

In conjunction with the production of test articles for preclinicalstudies, two batches of tegavivint suspension were prepared: one batchmade with sorbitol and one without sorbitol. Stability evaluation ofthese suspensions, stored at 5° C., 25° C./60% RH, and 40° C./75% RH,indicated that the suspensions were reasonably stable at all conditions.

A portion of the vials from the pig-study batch was autoclaved at 121°C. for 20 minutes using a liquid cycle and the formulation, designatedas batch 515-76 and FID5910.

Stability data indicated that autoclaving had no significant effect ondegradation but did appear to increase particle size.

Example 13 Engineering Studies of Nanosuspension

Several engineering batches of nanosuspension of tegavivint wereprepared in anticipation of clinical manufacture, and certainalterations to the process were necessary to maintain compliance andminimize loss and contamination. Partly for reasons of safety and partlyto incorporate a larger-surface-area filter for extraction purposes, theglass pressure funnel that had been used for extraction was replacedwith a stainless steel inline filter housing fitted with a 55-umstainless steel filter element (Pall).

Pressurization of the extraction apparatus, which had previously usednitrogen, was performed using a peristaltic pump, as this pump was alsoto be incorporated into the process as a means by which to sterilefilter both the diluent and dispersant rather than to autoclave them. Ametered peristaltic pump unit was employed for filling the vials. Giventhe tendency for autoclaving to increase the particle size of theformulation, the first engineering batch (RD4050-5) was submitted forgamma irradiation.

Stability results showed minimal degradation and stability similar toprevious batches.

TABLE 3 Tegavivint 25 mg/mL Engineering Lot Impurities > 0.1% Total BC-RRT 0.60 RRT RRT 1.08 RRT 1.11 RRT 1.17 RRT Impurities > 2059(Impurity 1) 0.95 (Impurity 2) (Impurity 3) (Impurity 4) 1.23 0.1%Unsterilized 103.3% 0.52 0.21 0.12 0.44 0.30 ND 1.6 Autoclave 103.6%0.53 0.21 0.12 0.46 0.32 ND 1.6 Gamma 101.5% 0.42 0.21 0.12 0.73 0.610.1 2.2 Irradiated

Based on preliminary particulate testing, the formulation, as processed,contained some larger particulate that did not appear to be populousenough to significantly affect laser diffraction particle-sizemeasurements, but was significant enough to affect USP<788> testing. Tomitigate the particulate, a “polishing” filter, with a porosity smallenough to retain larger particles, but not so small as to affecttegavivint assay, was proposed. However, previous attempts to filter thesuspension resulted in significant assay losses. Therefore, PallCorporation was contracted to assess some of their membranes fortegavivint nanosuspension filtration suitability.

Portions of a non-best-clean suspension were filtered using various47-mm membranes available from Pall, with a pressure feedback pumpingsystem that is used to determine how much material can be processedbefore filter clogging and failure. The following membrane types wereused, and the filtrate tested for PSD and assay:

TABLE 4 D90 Assay Membrane Material (nm) (% LC) None (unfiltered N/A 25692.7 control) HDC II, 10-micron Polypropylene 259 92.5 HDC II, 6-micronPolypropylene 253 92.2 Ultipleat ® Polypropylene 200 88.1 6-micronUltipleat ® Polypropylene 207 91.9 4.5-micron Glass-fiber filter Glassfiber Clogged immediately, not tested Depth filter Polypropylene Causedvisible clearing of filtrate, not tested Mini Profile ®, Polypropylene206 93.3 5-micron

The 6-micron HDCII membrane was chosen as the best candidate because itshowed no discernable impact on either the assay value or theparticle-size distribution of the nanosuspension. However, thecomparatively long lead-times of the Pall filters were a limiting factorin the timely production of clinical material. Therefore, an alternativewas sought, and Sartorius 8-micron polypropylene filters were used inthe manufacture of two engineering batches for use in determiningbioburden for sterilization validation purposes.

Unfortunately, the assay values of the two batches were negativelyaffected by the filtration (80.9% LC and 91.9% LC, respectively). Asthey were not representative of the final clinical batch profile, thebatches were discarded, and another engineering batch was processedusing a Pall HDC membrane filter. This batch assayed within 90 to 110%LC and the Pall HDC filter was incorporated into the final step of themanufacturing process prior to filling of the vials.

Example 14 Pharmacokinetic Study of Tegavivint Following SlowIntravenous Bolus Administration to Female Sprague-Dawley Rats

The objective of this study was to investigate the pharmacokinetics oftegavivint following a single intravenous slow bolus administration oftegavivint to female Sprague-Dawley rats.

The study was performed using parallel design (n=4/group) with serialsampling, as summarized in Table 5:

TABLE 5 Group No. of Dose Dose Volume Concentration Treatment NumberRats (mg/kg) (mL/kg) (mg/mL) BC2059 G1 4 10 2 5

Source

Sprague-Dawley rats used in the study were obtained from In-house animalresource facility, Advinus Therapeutics Ltd., Bengaluru, India. Theanimals were about 10-11 weeks of age on the day of dosing.

Identification

Each animal was identified with a unique identification number indicatedon the cage card and turmeric solution marking on the animal body. Thecage card identified each cage with study number, identification number,species and strain, dose and gender.

Housing and Environment

Rats were acclimatized to the study area conditions for 3 days beforedosing. Animals were housed (one per cage) in polypropylene cages andmaintained in controlled environmental conditions with 12 h light and 12h dark cycles. The temperature and humidity of the room was maintainedbetween 22±3° C. and 40-70%, respectively. The room underwent 10-15fresh air change cycles per hour.

Food and Water

The experimental animals were provided ad libitum of standard pelletedfood (Teklad Certified (2014C) Global 14% Protein Rodent MaintenanceDiet-Rodent pellet food, manufactured by Harlan Laboratories B.VMaasheseweg 87c PO Box 553, 5800, AN Venray, The Netherlands.

Dose Preparation and Administration

The stock formulation (25 mg/mL) was provided. Accurately, 600 μL ofdose formulation (Stock, 25 mg/mL) was transferred to labeled glasscontainer. To this, 2.4 mL of 5% dextrose solution was added, vortexmixed and sonicated to obtain homogeneous suspension of 5 mg/mLstrength. Animals were dosed under fed condition. Rats were administereda single dose of 10 mg/kg of tegavivint by slow intravenous bolus (over1.5 min) jugular vein catheter using 1 mL BD syringe guided with 23 Gblunt needle at a dose volume of 2 mL/kg. Syringes used for dosing wereweighed before and after dose administration in order to calculate theactual dose administered.

Sample Collection and Processing

Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 and24 h post dose. At each time point, approximately, 0.25 mL of blood waswithdrawn from jugular vein of the cannulated rat and transferred to alabeled microfuge tube containing 200 mM K₂EDTA (20 μL per mL of blood).Following sampling, equal volume of heparinized saline was replaced intothe catheter. The blood samples were kept on wet ice at all timesimmediately after collection and the plasma was separated bycentrifugation at 5000 g for 5 minutes at 4±2° C. The plasma sampleswere separated within 1 h of scheduled time and stored below −60° C.until bioanalysis.

Bioanalysis

Bioanalysis was performed using fit-for-purpose LC-MS/MS method for thequantification of BC2059 in rat plasma samples. The calibration curve(CC) for the method consisted of at least 6 non-zero calibrationstandards along with a blank and blank with internal standard sampleswith a lower limit of quantification (LLOQ) of 0.050 μg/mL. Studysamples were analyzed along with three sets of quality control samples(9 QC samples; low, medium and high QC samples in triplicate).

Pharmacokinetic Data Analysis

The pharmacokinetic parameters for tegavivint were calculated using thenon-compartmental analysis tool (extra vascular) of the validatedPhoenix® WinNonlin® software (version 6.3). The area under theconcentration time curve (AUClast and AUCinf) was calculated by lineartrapezoidal rule. The CO (back extrapolated concentration at time zero)was estimated following intravenous bolus dose administration byback-extrapolating the first two concentration values. The total plasmaclearance (CL) and volume of distribution at steady-state (Vss) wereestimated values. The elimination rate constant value (k) was calculatedby linear regression of the log-linear terminal phase of theconcentration-time profile using at least 3 declining concentrations interminal phase with a correlation coefficient of >0.8. The terminalhalf-life value (T½) was calculated using the equation 0.693/k. Thealpha and beta half-lifes were calculated and reported.

Experimental Results

Following single slow intravenous bolus administration of tegavivint(Dose: 10 mg/kg) to rats, the mean plasma clearance (CL) was estimatedto be 9.92 mL/min/kg, which is about 5.5-fold lower than the normal ratliver blood flow of 55 mL/min/kg. The mean plasma volume of distributionat steady state (Vss) was found to be almost 9.34-fold greater than thenormal body water of 0.7 L/kg, possibly suggesting wide distributioninto tissue compartments. The semi log plasma concentration-time plotsindicate that BC2059 exhibited bi-exponential elimination pattern withrapid distribution half-life (T½ alpha) of 0.546 h and long terminalplasma half-life (T½ beta) of 13.8 hours.

TABLE 6 PK Characteristics of BC2059 T_(1/2) T_(1/2) CL V_(ss) C₀AUC_(last) AUC_(inf) MRT_(last) Alpha Beta (mL/min/kg) (L/kg) (μg/mL)(μg · h/mL) (μg · h/mL) (h) (h) (h) 9.92 ± 6.54 ± 62.2 ± 14.6 ± 17.2 ±4.07 ± 0.546 ± 13.8 ± 1.79 3.07 7.13 3.69 3.29 0.579 0.0686 3.11

-   -   Regression points 0.5, 1 and 2 h for alpha phase and 6, 8, 12        and 24 h for terminal beta phase were selected to calculate        elimination rate constant.

Example 15 A Dose-Escalating Intravenous Infusion Study of Tegavivint inMale Beagle Dogs

-   -   Study Animals: Four male non-naïve Beagle dogs were released by        Xenometrics for study use on Apr. 20, 2017. The animals were fed        Harlan Teklad® Global 25% Protein Certified Dog Diet 2025C ad        libitum throughout the study (except for brief periods during        in-life procedures when it was    -   Dosing: Animals were dosed via intravenous (IV) infusion (via        jugular vein) for 4 hours (h) [±5 minutes (min)].

TABLE 7 Study Dosing Summary Infusion Concen- Rate Dose Dosing Dosetration (mL/kg/ Number Day Test Article (mg/kg) (mg/mL) hour) 1 and 2 1and 3 BC-2059 in 5 1 1.25 3 and 4 5 and 8 Poloxamer 188 10 2 1.25 5 and6 12 and 16 and sorbitol; 5% 15 2 1.88 dextrose as the diluent

Pharmacokinetic (PK) Blood Collection:

Blood samples were collected on the last dosing day (Dose 6; 15 mg/kg)prior to start of infusion and at 4, 12, 24, 36, 48, and 72 h after thestart of infusion. All blood samples were collected within 10 minutes ofthe target time and processed per protocol. Bioanalytical resultsindicated tegavivint was present in all plasma samples.

TABLE 8 Calculated Plasma BC-2059 Values (ng/mL) in Beagle DogsFollowing a 15 mg/kg 4 h IV Infusion Timepoint (h Postdose) Animal No.Predose 4 12 24 36 48 72 96 120 JY1001 404 1310 916.0 1120 834 663 511360 321 JY1002 309 1320 1050 1120 931 656 480 310 262 JY1003 284 1150777 1070 708 647 524 364 281 JY1004 257 1600 784 723 674 664 393 277 228

The pharmacokinetic parameters were determined for BC-2059 following thefinal 15 mg/kg infusion of the drug over 4 h. The mean values arepresented in Table 8 above. The data indicate a half-life of 53.0 h andan overall AUC_(0-120h) of 73480 ng*h/mL.

TABLE 9 Mean PK Parameters for BC-2059 Dose Cmax AUC_(0-120 h) T½(mg/kg) Route (ng/mL) (h*ng/mL) (h) 15 IV over 4 h 1345 ± 187 73480 ±5803 53.0 ± 6.7 n = 4 males

Example 16 Nebulizing Delivery of Tegavivint Formulations

The purpose of this experiment was to test nebulized delivery ofnanosuspensions. This experiment demonstrated that nebulized deliverywas successful.

Tegavivint particles suspended in Poloxamer 188/sorbitol at aconcentration of 25 mg/mL were used.

These formulations were applied to the mice in the form of aerosols,through the method of whole body exposure. The mice were placed inside aplastic box. This box was sealed and connected by one of its sides tothe outlet of the nebulizer device, and on the other side to a system ofclosed water. The whole procedure was carried out inside the fume hoodof the animal room.

For the first experiment, the nebulizer kit of SATER LABS was used. Thisdevice uses the jet system. The device was primed with 5 ml of the drug,i.e. 125 mg of tegavivint (BC2059), for each group of 5 mice, and thenthe device was connected to the power source for nebulization. Theenergy was supplied by a DeVilbiss compressor model 646, which allows5-7 pounds of pressure, and a flow of 6-8 liters per minute. For thesecond experiment, the device used was the Altera, ultrasonic nebulizer.

For the two experiments, 10 male bcat-Ex3 mice were used for each set.These mice were separated into 2 groups of 5 mice each. The first groupreceived the drug daily, for 5 consecutive days. The second groupreceived the drug only once (the fifth day). On day 5 all mice weresacrificed, lung harvested, and samples were stored at −30 degrees, intwo labeled nylon bags, each containing the 5 samples from each group.

Results:

TABLE 10 Matrix/ Lung Lung Group Animal Bleed ConcentrationConcentration Label ID ID time Analyte (ng/mL) (ng/g) Aerosol 1 Day #1-12 1 Mouse BC2059 14200 56800 Oct. 10, 2017 lung Aerosol 1 Day #2-2 2 2Mouse BC2059 1300 5200 Oct. 10, 2017 lung Aerosol 1 Day #3-3 2 3 MouseBC2059 4270 17100 Oct. 10, 2017 lung Aerosol 1 Day #4-4 2 4 Mouse BC20591260 5040 Oct. 10, 2017 lung Aerosol 1 Day #5-5 2 5 Mouse BC2059 419016800 Oct. 10, 2017 lung Aerosol 5 Day #1-6 3 6 Mouse BC2059 4640 18600Oct. 10, 2017 lung Aerosol 5 Day #2-7 3 7 Mouse BC2059 1650 6600 Oct.10, 2017 lung Aerosol 5 Day #3-8 3 8 Mouse BC2059 3620 14500 Oct. 10,2017 lung Aerosol 5 Day #4-9 3 9 Mouse BC2059 3080 12300 Oct. 10, 2017lung Aerosol 5 Day #5-10 3 10 Mouse BC2059 3550 14200 Oct. 10, 2017 lungNebulizer 1 Day #1-11 4 11 Mouse BC2059 2840 11400 Oct. 10, 2017 lungNebulizer 1 Day #2-12 4 12 Mouse BC2059 2910 11600 Oct. 10, 2017 lungNebulizer 1 Day #3-13 4 13 Mouse BC2059 8660 34600 Oct. 10, 2017 lungNebulizer 1 Day #4-14 4 14 Mouse BC2059 3780 15100 Oct. 10, 2017 lungNebulizer 1 Day #5-15 4 15 Mouse BC2059 601 2400 Oct. 10, 2017 lungNebulizer 5 Day #1-16 5 16 Mouse BC2059 4770 19100 Oct. 10, 2017 lungNebulizer 5 Day #2-17 5 17 Mouse BC2059 4290 17200 Oct. 10, 2017 lungNebulizer 5 Day #3-18 5 18 Mouse BC2059 4690 18800 Oct. 10, 2017 lungNebulizer 5 Day #4-19 5 19 Mouse BC2059 3040 12200 Oct. 10, 2017 lungNebulizer 5 Day #5-20 5 20 Mouse BC2059 5730 22900 Oct. 10, 2017 lungLLOQ: 20.0 ng/g “Aerosol” refers to standard aerosol jet nebulizer(Safer Labs); “Nebulizer” refers to nebulizer ultrasonic eRapid machine(Altera) “1 Day” refers to single-dose on Day 5; “5 Day” refers to 5daily doses on Days 1-5.

Example 17

Pig Studies with Liquid Formulation of Tegavivint and Nanosuspension ofTegavivint Liquid Suspension was Poorly Tolerated by Pigs

Tegavivint was intravenously administered to minipigs in a series ofpharmacokinetic studies to determine a formulation that would besuitable for GLP toxicology studies, both in terms of systemic exposuresand in tolerability to the drug product formulation. These studies wereall conducted at Sinclair Research (Auxvasse, Mo.).

In the first study, the drug was obtained in a formulation consisting ofTween 80, ethanol, polyethylene glycol (PEG) and vitamin E TGPS (d-alphatocophenyl polyethylene glycol 1000 succinate). This stock formulationwas diluted in 20% Intralipid® (phospholipid stabilized soybean oil) tothe final dose concentration. Two pigs were administered 1.7 mg/kg over6 h and two pigs were administered 2.2 mg/kg over 24 h. The formulationprovided good systemic exposures.

Comparing dose normalized values the shorter 6 h duration, relative tothe 24 h duration, resulted in higher peak concentrations (C_(max)/Dose)at the end of the infusion as the total dose was given over a shorterduration. However, the overall systemic exposures over time (AUCs/Dose)were similar between the two infusion durations, meaning that with alonger infusion time, it was possible to obtain similar overall systemicexposures while avoiding higher peak plasma concentrations.

However, while good systemic exposures were observed with thisformulation, marked infusion reactions were observed, and thisformulation was not tolerated by minipigs either over a 6 h or a 24 hinfusion period. It was hypothesized that the tween/ethanol/PEG/VitaminE/Intralipid solvent-based excipients and probable drug precipitationwere responsible for the infusion reactions and not tegavivint itself.Indeed, tegavivint does not cause hemolysis of red blood cells eitherwhen in the nanoparticle form or when dissolved in DMSO.

Lyophilized Nanosuspensions of Tegavivint were Better Tolerated butUltimately were Abandoned Due to Stability Issues

In the subsequent study, tegavivint was milled to a nanoparticle sizeand a non-solvent formulation was used. In this study, Study B01-109, alyophilized form of tegavivint was obtained and reconstituted in waterto provide a stock formulation consisting of a suspension of tegavivint10 mg/mL, 2.5 mg/mL Poloxamer 188 and 5 mg/mL sorbitol. This stocksolution was diluted with normal saline to the final requisiteconcentrations for intravenous administration. Two pigs were infusedwith 2.9 mg/kg and 2 pigs were infused with 12.3 mg/kg over a 4 hinfusion. One pig in the 12.1 mg/kg dose group had very high systemicexposures. Notwithstanding this pig, dose normalized AUC, and to alesser extent the C_(max), were dose linear across the 2.8 to 12.1 mg/kgdose given over the same duration. With the exception of the one pig inthe 12.1 mg/kg dose group, the dose normalized exposures were less withthis lyophilized form of nano-milled tegavivint compared to thetween/ethanol/PEG/Vitamin E/Intralipid solvent-based formulation used inStudy B01-107.

Nevertheless, as this formulation was well-tolerated by the minipigswith none of the infusion reactions observed in Study B01-107, thelyophilization process was scaled up for future work. However, in thescale-up process, we were unable to obtain a lyophilized product withadequate stability and an alternative lyophilized formulation oftegavivint was needed.

In Study TXPK-006-2059-24 h, a lyophilized formulation of milledtegavivint was used. The lyophilized formulation used was reconstitutedin water to final concentrations of BC2059 25 mg/mL, 0.125%polyvinylpyrrolidone (PVP), 0.0625% NaDeoxycholate (NaDOC) and 10%sucrose. This bulk solution was diluted in normal saline to therequisite concentrations for dose administration to two pigs pertreatment group at 12.3 or 49.2 mg/kg over a 24 h infusion. Test articleflocculation was observed in the syringes and the syringes were agitatedthroughout the 24 h infusion period. Nonetheless, systemic exposureswere exceedingly low compared to Studies B01-107 and B01-109. Subsequentformulation work showed that saline with this lyophilized formulationresulted in test article aggregation and an ionic (saline) diluent couldnot be used.

Frozen Liquid Nanosuspensions of Tegavivint Worked

At this point, lyophilization was abandoned and frozen liquidformulations of the milled tegavivint were investigated. In StudyTXPK-001-2059-pig 24 h PK, three minipigs were assigned one per group toone of three groups with the formulations administered over 24 h. Twofrozen milled suspensions were provided by Particle Sciences. BC2059-1was a 25 mg/mL BC2059, 0.125% PVP, 0.0625% NaDOC, 10% sucrose,suspension (Batch no. 515-10) and BC2059-2 was a 25 mg/mL BC2059, 0.625%poloxamer 188, 10% sorbitol, suspension (Batch no. 515-13). The diluentfor both of these formulations was D5W. A third group was the BC2059PVP/NaDOC/sucrose frozen formulation BC2059-1 with a poloxamer188/saline diluent to investigate a possible role for the poloxamer 188in systemic exposures.

Of these 3 frozen test article formulations, the 25 mg/mL tegavivint,0.625% poloxamer 188, 10% sorbitol, nanosuspension diluted with D5Wshowed the highest systemic exposures. The dose normalized AUC for thisformulation was somewhat less than the dose normalized exposuresobserved in the 24 h infusions in Study B01-107, but not markedly so.The dose normalized exposures were considerably higher than observed in3 of 4 pigs in Study B01-109, indicating that the saline diluent in thatstudy might have affected systemic exposures with the lyophilizedpoloxamer 188 formulation of BC2059, albeit to a much lesser extent thanobserved with the PVP/NaDOC formulation of tegavivint.

TABLE 11 Single-Dose Pharmacokinetics of BC2059 in the MinipigFormulation Actual and Infusion Dose Cmax Cmax/ AUC AUC/ Study Duration(mg/kg) (ng/mL) Dose (ng*h/mL) Dose B01-107 BC2059-13^(a) 1.7 1800, 940 1059, 553  7960, 5395 4682, 3174 6h BC2059-13^(a) 2.2  270, 1120 123,509  5544, 12889 2520, 5859 24 h B01-109 BC2059^(b) 2.8 448, 540 160,193 2144, 2417 766, 863 4 h BC2059^(b) 12.1 26100, 4690  2157, 388 28778, 11617 2378, 960  4 h TXPK-006- BC2059^(c) 12.3 30, 23 3, 2 506,360 43, 31 2509-24 h 24 h pig tol BC2059^(c) 49.2  91, 106 2, 2 1526,1719 31, 35 24 h TXPK-001- BC2059-1^(d), 47 2860 61 16269 346 2059-pig24 24 h PK BC2059-2^(e), 53 4370 82 78893 1489  24 h BC2059-3^(f), 512590 41 49645 973 24 h ^(a)20 mg/mL BC2059 in 30% Ethanol, 50% PG, 10%Tween 80, and 10% D-α-Tocopherol polyethylene glycol 1000 succinate (LotP492-01); values reported for 2 minipigs ^(b)10 mg/mL BC2059, 2.5 mg/mLPoloxamer 188 and 5 mg/mL sorbitol (Lot No. BET 1213-001-29); valuesreported for 2 minipigs ^(c)25 mg/mL BC2059, 0.125% PVP, 0.0625% NaDOCand 10% sucrose (Lot No. BET 1213-001-49); values reported for 2minipigs ^(d)25 mg/mL BC2059, 0.125% PVP, 0.0625% NaDOC, 10% sucrose,nanosuspension (Lot 515-10) diluted to 2 mg/mL final concentration indextrose 5%; single minipig ^(e)25 mg/mL BC2059, 0.625% poloxamer 188,10% sorbitol, nanosuspension (Lot 515-13) diluted to 2 mg/mL finalconcentration in dextrose 5%; single minipig ^(f)25 mg/mL BC2059, 0.125%PVP, 0.0625% NaDOC, 10% sucrose, nanosuspension (Lot 515-10) diluted to2 mg/mL final concentration in poloxamer 188/saline to a finalconcentration of 0.05% poloxamer; single minipig

Based on the results of these single dose infusion studies in minipigs,the formulation selected for repeated 2-dose toxicology studies was thefrozen formulation of 25 mg/mL BC2059 with 0.625% poloxamer 188, 10%sorbitol (nanosuspension) and diluted with D5W

During the conduct of the 2-dose non-GLP studies to support doseselection for the IND-enabling GLP toxicology studies, we learned in thescale-up process and production of Particle Sciences Batch no. 515-33that freezing of the formulation resulted in aggregation of tegavivintin the vials.

Given this aggregation with freezing, we subsequently made the decisionto pursue the 25 mg/mL nano-milled BC2059 in 0.625% poloxamer 188, 10%sorbitol formulation holding at 2-4° C. Aggregation was not observed inmultiple lots of the milled BC2059 suspension, provided the formulationwas not frozen. The poloxamer/sorbitol formulation, refrigerated at 2-4°C., was used for the IND-enabling GLP toxicology studies and in thenon-GLP beagle dog study.

Example 18 Efficacy of Nebulized Tegavivint in a Mouse Model ofIdiopathic Pulmonary Fibrosis

The purpose of this experiment was to investigate tegavivintnanosuspension in a mouse model of bleomycin-induced idiopathicpulmonary fibrosis (IPF). Test articles were as follows:

Tegavivint (BC2059) in a nano-milled suspension 25 mg/mL in 0.625%poloxamer 188 and 10% sorbitol. The test article was refrigerated atabout 4° C.

Nebulizing equipment was Altera ultrasonic eRapid machine nebulizer(model #678G1002).

Animals were 8-12 week old C57BL/6 male mice (Jackson Lab, Bar Harbor,Me.).

Experimental Procedure

TABLE 12 Group # of mice Day 0 Day 5-21 1 4 IT PBS 50 μl 5 ml of Vehicle(0.625% poloxamer 188/10% sorbitol) aerosol, BID 2 4 IT Bleomycin 5 mlof Vehicle 0.025 U in 50 μl (0.625% poloxamer saline 188/10% sorbitol)aerosol, BID 3 5 IT Bleomycin 5 ml of 25 mg/ml 0.025 U in 50 μltegavivint aerosol, saline BID

Murine model of pulmonary fibrosis was induced by intratracheally (IT)injected bleomycin (APP Pharmaceuticals, Schaumburg, Ill.). One dose of0.025 U bleomycin dissolved in 50 microliters of Saline 0.9%, or PBS ascontrol was administered to each animal on day 0.

Tegavivint nanosuspension was applied to Group 3 in the form ofaerosols, through the method of whole body exposure. The mice wereplaced inside a plastic box. This box was sealed and connected by one ofits sides to the outlet of the nebulizer device, and on the other sideto a system of closed water. The whole procedure was carried out insidethe fume hood of the animal room. In each treatment session, 5 ml of 25mg/ml Tegavivint (125 mg) was nebulized over 15 min to each group of 4-5mice in the chamber. To increase exposure of the mice to the aerosol,Tegavivint that precipitated in the aerosol chamber was collected with asyringe and re-nebulized a second and a third time. Mice were nebulizedtwice a day between day 5 and day 21 after administration of bleomycin.Group 1 and 2 received nebulized vehicle 5 ml in the same manner.

The body weight of animals was recorded on Days 0, 5, 8, 12, 16, 19, and21.

Measurements of lung mechanics were performed on Day 21 as previouslydescribed (Morales-Nebreda L, et al. AJRCMB 2015) on Day 21 using aFlexiVent mouse ventilator (Scireq, Montreal, PQ, Canada) according tothe protocols established by Scireq. A standard ventilation history foreach mouse was obtained with three total lung capacity maneuvers beforethe forced oscillation and quasistatic pressure-volume curve protocolsthat were used to calculate airway resistance, dynamic and quasistatictissue compliance, and elastance.

On Day 21 all animals were sacrificed and lungs were harvested. Totallung collagen content was evaluated using the Hydroxyproline Assay aspreviously described (Morales-Nebreda L, et al. AJRCMB 2015). In brief,mouse lungs were harvested and suspended in 1 ml of 0.5 M acetic acidand then homogenized, first with a tissue homogenizer (60 s on ice) andthen using 15 strokes in a Dounce homogenizer (on ice). The resultinghomogenate was spun (12,000×g) for 10 minutes, and the supernatant wasused for subsequent analyses. Collagen standards were prepared in 0.5 Macetic acid using rat tail collagen (Sigma-Aldrich, St. Louis, Mo.).Picrosirius red dye was prepared by mixing 0.2 g of Sirius red F3B(Sigma-Aldrich) with 200 ml of water; 1 ml of the Picrosirius red dyewas added to 100 μl of the collagen standard or the lung homogenates andthen mixed continuously at room temperature on an orbital shaker for 30minutes. The precipitated collagen was then pelleted and washed oncewith 0.5 M acetic acid (12,000×g for 15 min each). The resulting pelletwas resuspended in 1 ml of 0.5 M NaOH and Sirius red staining wasquantified spectrophotometrically (540 nm) using a colorimetric platereader (Bio-Rad, Hercules, Calif.).

Results

Group 2 showed statistically significant body weight reduction afterbleomycin treatment, which is one of the indicators of IPF induction. Incontrast, inhaled tegavivint treatment in Group 3 reversed the bodyweight loss caused by the bleomycin induced lung injury.

TABLE 13 Change in body weight (%) Animal # Group 1 Group 2 Group 3 13.24 −10.85 −2.83 2 9.13 −1.25 5.2 3 9.85 −4.67 7.3 4 12 −2.62 7.3 5 7.9

Further, bleomycin induced decreased lung compliance in Group 2, whichindicates the induction of fibrosis. Inhaled tegavivint treatment afterbleomycin injury in Group 3 reversed the compliance values to near thoseof the sham-treated controls in Group 1.

TABLE 14 Compliance (ml/cm H₂0) Animal # Group 1 Group 2 Group 3 10.076337 0.044172 0.055153 2 0.068275 0.042324 0.056036 3 0.0580570.048667 0.067618 4 0.07324 0.042422 0.054295 5 0.056101

Further, the total lung collagen content as measured by theHydroxyproline assay showed marked increase in Group 2, indicatingactive fibrosis after bleomycin injury; in contrast, inhaled tegavivinttreatment after bleomycin injury in Group 3 reversed this change and thecollagen levels are closed to sham-treated controls in Group 1.

TABLE 15 Hydroxyproline (μg/half lung) Animal # Group 1 Group 2 Group 31 51.296 75.632 70.016 2 36.32 85.824 39.44 3 44.432 68.768 45.68 437.568 77.504 58.784 5 64.296

Thus, this experiment demonstrated that tegavivint has a great potentialto treat IPF.

Example 19 Assessing Aerosolized Tegavivint Formulations

A series of BC-2059 (tegavivint) formulations were aerosolized usingvibrating mesh and compressed air nebulizers to determine the mostefficient method of aerosol generation. The aerosols were characterizedfor aerosol concentration and particle size distribution in a rodentnose-only exposure chamber. Each formulation had different variablesadjusted to assess impact on aerosol performance. These includedparticle size reduction of the API, excipient profile and nebulizerutilized.

The objective of this study was to determine a method by which testarticle BC-2059 could be aerosolized for inhalation studies for rodents.

Test article BC-2059 was suspended in 0.1% Tween 80 in purified water ata concentration of 15 mg/mL. The suspension was sonicated using aCovaris S220x Ultrasonicator (Covaris, Boston Mass.) and then mixed on avortex for one minute. Sonication and mixing by vortex was repeated atotal of 15 times.

The remaining bulk BC-2059 powder was ground in a Planetary Ball Mill(Retsch, Germany) for 10 minutes at 150 RPMs utilizing a 12 mL ball milljar and three metal balls. The milled BC-2059 powder was suspended in0.1% Tween 80 in purified water at a concentration of 15 mg/mL. Thesuspension was sonicated using the procedure outlined above.

The remaining bulk BC-2059 powder was ground in a Planetary Ball Mill(Retsch, Germany) for 60 minutes at 300 RPMs utilizing a 12 mL ball milljar and three metal balls. The milled BC-2059 powder was suspended in0.1% Tween 80 in purified water and 10% PEG 400 in purified water at aconcentration of 15 mg/mL. The suspensions were sonicated using theprocedure outlined above. An additional 15 mg/mL BC-2059 suspensionusing 10% ethanol in purified water was prepared. The suspension wassonicated for 10 minutes using a VWR sonicator (VWR, Radnor Pa.) andmixed for 4 minutes using a vortex mixer.

An additional formulation (nanomilled suspension of 25 mg/mL BC-2059 in0.625% poloxamer 188 and 10% sorbitol) was used as received withoutfurther modification.

The aerosols were generated from formulations prepared with a series ofsurfactants with 4 separate nebulizers (Aeroneb Solo (Aerogen, Ireland),Pari LC Plus (Pari Respiratory Equipment Inc. Midlothian Va.), HospitakUp Mist, Hospitak Inc. Farmdale, N.Y.), and Hudson Micro-Mist (TeleflexInc. Research Triangle Park, N.C.) and transitioned into a 2 tierflow-past rodent exposure system.

The total concentration of aerosol in the exposure atmosphere wasdetermined by the analysis of filter samples (GF/A 47-mm filters).Filter samples were collected at a nominal flow rate of 0.3 L/min.Filter samples collected throughout the study were analyzedgravimetrically to determine the total aerosol concentration, andsubmitted for HPLC analysis.

Filters with test article were extracted in 1:1 acetonitrile: methanoland analyzed by the HPLC-UV assay.

Particle size distribution (PSD) of the test article was measured at thebreathing zone using an In Tox, mercer style cascade impactor.

Results

The aerosol concentrations (gravimetric and chemical) are shown in Table16 below.

TABLE 16 Method Development Summary Formula- Total BC-2059 API tionAerosol Aerosol PSD Conc. Conc. Conc. Reduction Excipients (mg/mL)Nebulizer (mg/L) (μg/L) Covaris 0.1% 15.04 Aeroneb 0.247 1.3 Tween 80Solo Covaris and 0.1% 15.06 Hospitak 0.229 66 Ball Mill Tween 80 (60min) Covaris and 0.1% 15.58 Pari LC 0.123 N/A Ball Mill Tween 80 Plus(10 min) Covaris and 0.1% 15.08 Hudson 0.093 86 Ball Mill Tween 80MicroMist (60 min) Sonicator and 10% 15.00 Hudson 0.027 20.2 Ball MillEthanol MicroMist (60 min) Covaris and 10% 15.08 Hudson 2.58 7.2 BallMill PEG-400 MicroMist (60 min) Tegavivint 0.625% 25.0 Hudson 2.47 484BC2059 poloxamer MicroMist nano-milled 188/10% suspension sorbitol

Particle size for test atmospheres was measured using an In-Tox Cascadeimpactor for suspensions prepared with 0.1% Tween 80 in ultrapure water,and the sponsor provided poloxamer suspension using a compressed airnebulizer. The mass median aerodynamic diameter and geometric standarddeviation for each formulation are listed in Table 17 below. Particlesize distributions are shown in FIG. 1 and FIG. 2.

TABLE 17 Particle Size Distribution API PSD Formulation ReductionExcipients Conc. (mg/mL) Nebulizer MMAD GSD R² Covaris and Ball 0.1%Tween 80 15.06 Hospitak 1.26 2.21 0.99 Mill (60 min) Tegavivint 0.625%25.0 Hudson 2.46 1.45 0.98 BC2059 nono- poloxamer μmist milled 188/10%suspension sorbitol

CONCLUSION

Formulations of BC-2059 were nebulized and introduced to nose-onlyinhalation exposure chamber. The exposure atmospheres were characterizedfor aerosol concentration using gravimetric and HPLC assay. The highestgravimetric aerosol concentration was measured at 2.47 mg/L for thepoloxamer formulation, which corresponded to 0.48 mg/L of active testarticle. The particle size distribution for this formulation wasmeasured by cascade impactor and had a MMAD of 2.46 μm with a geometricstandard deviation of 1.45 μm.

In reviewing the poloxamer formulation results against the previousresults, the aerosol concentration of 0.484 mg/L of BC2059 would resultin a 1.5 mg/kg pulmonary deposited dose (10% DF) to a 30 gram mouse in30 minutes. Based on standard mouse lung weights, this would result in0.2 mg/g in the lung tissue. The previous testing resulted in 0.02 mg/g(assayed concentration).

Therefore, this BC2059 nanomilled suspension which was nebulized gavethe most optimal concentration in the aerosol in comparison to the otherBC2059 formulations.

What is claimed is:
 1. A composition comprising: a) particles of acompound of formula I

wherein R_(A) is hydrogen, R₇ and R₈ are independently selected from Hand SO₂NR₃R₄, wherein one of R₇ and R₈ is hydrogen and wherein NR₁R₂ andNR₃R₄ are independently 6- to 15-membered heterocycloalkyl containingone nitrogen in the ring, or a pharmaceutically acceptable salt, ester,amide, stereoisomer or geometric isomer thereof; and b) a surfactant;wherein the particles have an effective D50 of less than or equal to 500nm and D90 of less than or equal to 1.0 micrometer (μm) when measuredusing laser diffraction.
 2. The composition of claim 1, wherein thecompound of Formula I is tegavivint or a pharmaceutically acceptablesalt, ester, amide, stereoisomer or geometric isomer thereof.
 3. Thecomposition of claim 1, wherein the composition is a nanoparticulatecomposition.
 4. The composition of claim 1, wherein the surfactant is apoloxamer surfactant.
 5. The composition of claim 1 wherein thepoloxamer surfactant is Poloxamer
 188. 6. The composition of claim 1wherein the composition further comprises a stabilizer.
 7. Thecomposition of claim 6, wherein the stabilizer is selected from thegroup consisting of a sugar, a polyol, a polysorbate surfactant andpolyvinylpyrrolidone (PVP).
 8. The composition of claim 7, wherein thesugar is selected from the group consisting of sucrose and trehalose. 9.The composition of claim 7, wherein the polyol comprises sorbitol andmannitol.
 10. The composition of claim 1, wherein the concentration ofthe compound is between about 10 mg/ml and about 25 mg/ml.
 11. Thecomposition of claim 1, wherein the concentration of the compound isabout 25 mg/ml.
 12. A composition comprising: a. 10-25 mg/ml oftegavivint or a pharmaceutically acceptable salt, ester, amide,stereoisomer or geometric isomer thereof; b. Poloxamer 188; and c.sorbitol; wherein tegavivint or the pharmaceutically acceptable salt,ester, amide, stereoisomer or geometric isomer thereof is in the form ofa nanosuspension comprising particles of tegavivint or thepharmaceutically acceptable salt, ester, amide, stereoisomer orgeometric isomer thereof, and wherein the particles have an effectiveD50 of less than or equal to 500 nm and D90 of less than or equal to 1.0micrometer (μm) when measured using laser diffraction.
 13. Thecomposition of claim 12, wherein the amount of tegavivint or thepharmaceutically acceptable salt, ester, amide, stereoisomer orgeometric isomer thereof is 25 mg/ml; the amount of Poloxamer 188 is0.625%; and the amount of sorbitol is 10%, wherein the percentages areby weight of the composition.
 14. The composition of claim 1, whereinsaid composition is prepared by milling.
 15. The composition of claim 1,wherein said composition is prepared by LyoCell technology.
 16. Aprocess of preparing a composition comprising: a) mixing particles ofthe compound of formula I

wherein R_(A) is hydrogen, R₇ and R₈ are independently selected from Hand SO₂NR₃R₄, wherein one of R₇ and R₈ is hydrogen and wherein NR₁R₂ andNR₃R₄ are independently 6- to 15-membered heterocycloalkyl containingone nitrogen in the ring, or a pharmaceutically acceptable salt, ester,amide, stereoisomer or geometric isomer thereof; with a surfactant andan acceptable carrier to produce a suspension; b) roller milling orusing a high energy mill to mill the suspension of step (a); and c)adding a polyol to the particles of step (b).
 17. The composition ofclaim 16, wherein the composition exhibits long term stability.
 18. Thecomposition of claim 16, wherein the compound of formula I istegavivint, or a pharmaceutically acceptable salt, ester, amide,stereoisomer or geometric isomer thereof.
 20. The composition of claim1, wherein the composition is formulated: (a) into a dosage formselected from the group consisting of tablets, and capsules; (b) into adosage form selected from the group consisting of controlled releaseformulations, fast melt formulations, delayed release formulations,extended release formulations, pulsatile release formulations, and mixedimmediate release and controlled release formulations; (c) into a dosageform suitable for inhalation or parenteral administration, includingintramuscular, subcutaneous, intravenous and intradermal injection; or(d) any combination of (a), (b), and (c).
 21. A method of preventing,treating or ameliorating cancer or tumor metastasis in a mammal in needthereof comprising administering to said mammal an effective amount ofthe composition of claim
 1. 22. A method for treating cancer comprisingadministering to a subject in need thereof a combination of: 1) apharmaceutically effective amount of the nanoparticulate composition ofclaim 1; and 2) a pharmaceutically effective amount of at least oneadditional anti-cancer agent.
 23. The method of claim 22, wherein theadditional anti-cancer agent is selected from the group consisting ofantimitotic agents, antimetabolite agents, HDAC inhibitors, proteosomeinhibitors, immunotherapeutic agents, FLT-3 EGFR, MEK, PI3K and otherprotein kinase inhibitors, LSD1 inhibitors, and WNT pathway inhibitors,alkylating agents and DNA repair pathway inhibitors, anti-hormonalagents, anti-cancer antibodies, and other cytotoxic chemotherapy agents.24. A method of treating and/or preventing a fibrotic disease in amammal in need thereof comprising administering to said mammal aneffective amount of the composition of claim
 1. 25. The method of claim24, wherein the fibrotic disease is selected from the group consistingof pulmonary fibrosis, Dupuytren's contracture, scleroderma, systemicsclerosis, scleroderma-like disorders, sine scleroderma, livercirrhosis, interstitial pulmonary fibrosis, keloids, chronic kidneydisease, chronic graft rejection, and other scarring/wound healingabnormalities, post-operative adhesions, reactive fibrosis.